Enhancing the activity of zearalenone lactone hydrolase toward the more toxic α-zearalanol via a single-point mutation.

Zearalenone (ZEN) and its derivatives are estrogenic mycotoxins known to pose significant health threats to humans and animals. Especially, the derivative α-zearalanol (α-ZAL) is over 10 times more toxic than ZEN. Simultaneous degradation of ZEN and its derivatives, especially α-ZAL, using ZEN lactone hydrolases (ZHDs) is a promising solution to eliminate their potential hazards to food safety. However, most available ZHDs exhibit limited activity toward the more toxic α-ZAL compared to ZEN. Here, we identified a broad-substrate spectrum ZHD, named ZHDAY3, from Exophiala aquamarina CBS 119918, which could not only efficiently degrade ZEN but also exhibited 73% relative activity toward α-ZAL. Through rational design, we obtained the ZHDAY3(N153H) mutant, which exhibited the highest specific activity (253.3 ± 4.3 U/mg) reported so far for degrading α-ZAL. Molecular docking, structural comparative analysis, and kinetic analysis collectively suggested that the shorter distance between the side chain of the catalytic residue His242 and the lactone bond of α-ZAL and the increased binding affinity to the substrate were mainly responsible for the improved catalytic activity of ZHDAY3(N153H) mutant. This mechanism was further validated through additional molecular docking of 18 mutants and experimental verification of six mutants.IMPORTANCEThe mycotoxins zearalenone (ZEN) and its derivatives pose a significant threat to food safety. Here, we present a highly promising ZEN lactone hydrolase (ZHD), ZHDAY3, which is capable of efficiently degrading both ZEN and the more toxic derivative α-ZAL. Next, the ZHDAY3(N153H) mutant obtained by single-point mutation exhibited the highest specific activity for degrading α-ZAL reported thus far. We further elucidated the molecular mechanisms underlying the enhanced hydrolytic activity of ZHDAY3(N153H) toward α-ZAL. These findings represent the first investigation on the molecular mechanism of ZHDs against α-ZAL and are expected to provide a significant reference for further rational engineering of ZHDs, which will ultimately contribute to addressing the health risks and food safety issues posed by ZEN-like mycotoxins.

Engineering polysaccharide hydrolases in the product-releasing cleft to alter their product profiles.

Polysaccharide hydrolases are enzymes capable of hydrolyzing polysaccharides to generate oligosaccharides that have diverse applications in the food, feed and pharmaceutical industries. However, the detailed mechanisms governing the compositions of their hydrolysates remain poorly understood. Previously, we identified a novel neopullulase Amy117, which exclusively converts pullulan to panose by specifically cleaving α-1,4-glycosidic bonds. Yet, several enzymes with high homology to Amy117 produce a mixture of glucose, maltose and panose during pullulan hydrolysis. To explore this particular phenomenon, we compared the sequences and structures between Amy117 and the maltose amylase ThMA, and identified a specific residue Thr299 in Amy117 (equivalent to His294 in ThMA) within the product-releasing cleft of Amy117, which might be responsible for this characteristic feature. Using structure-based rational design, we have successfully converted the product profiles of pullulan hydrolysates between Amy117 and ThMA by simply altering this key residue. Molecular docking analysis indicated that the key residue at the product-releasing outlet altered the product profile by affecting the panose release rate. Moreover, we modeled the long-chain pullulan substrate G8 to examine its potential conformations and found that G8 might undergo a conformational change in the narrow cleft that allows the Amy117 variant to specifically recognize α-1,6-glycosidic bonds.

The N-terminal hydrophobicity modulates a distal structural domain conformation of zearalenone lacton hydrolase and its application in protein engineering.

Zearalenone (ZEN) is one of the most common mycotoxins in maize, wheat, barley, sorghum, rye and other grains. ZEN contamination in feed is an international health issue due to its estrogenicity by competitively binding to estrogen receptors. Enzymatic detoxification of ZEN is superior to physical and chemical methods in terms of safety, environmental impact and preserving nutritional value and palatability, but is hampered by both the currently limited repertoire of detoxifying enzymes and the lack of knowledge about their structure-function relationships. In this study, a ZEN lacton hydrolase candidate (ZHD11C) was identified from thermo-tolerant Fonsecaea multimorphosa CBS 102226, and characterized to be more thermostable than these reported homologues. An intriguing feature of ZHD11C is that the N-terminal hydrophobicity affects its thermal stability and causes conformational change of a domain far from the N-terminal. This finding was successfully applied to enhance the thermostability of the most active ZEN lacton hydrolase ZHD518 through rationally tailoring its N-terminal hydrophobicity. Our results not only provide more insights into the structure-function relationships of ZEN lacton hydrolases, but generate better candidate for bio-decontamination of zearalenone in feed industries.

Extracellular Volume Fraction Based on Cardiac Magnetic Resonance T1 Mapping: An Effective Way to Evaluate Cardiac Injury Caused by Cardiac Amyloidosis in Patients with Multiple Myeloma.

Multiple myeloma (MM) is a hematological malignancy of plasma cell origin. Cardiac amyloidosis (CA) is a common form of heart damage caused by MM and is associated with a poor prognosis. This study was a prospective cohort study and was aimed at evaluating the clinical predictive value of extracellular volume fraction (ECV) based on cardiovascular magnetic resonance (CMR) T1 mapping for cardiac amyloidosis and cardiac dysfunction in MM patients. Fifty-one newly diagnosed MM patients in Zhongnan Hospital of Wuhan University were enrolled in the study. A total of 19 patients (19/51; 37.25%) developed CA. The basal ECV of CA group was significantly higher than that of the non-CA group (p < 0.01). Multivariate logistic regression analysis showed that basal ECV (OR = 1.551, 95% CI 1.084-2.219, p < 0.05) and LDH1 level (OR = 1.150, 95% CI 1.010-1.310, p < 0.05) were two independent risk factors for CA. Further study demonstrated that basal ECV in the heart failure group was significantly higher than that of the nonheart failure group (p < 0.01). Notably, ROC curve showed that basal ECV had a good predictive value for CA and heart failure, with AUC of 0.911 and 0.893 (all p < 0.01), and the best cutoff values of 38.35 and 37.45, respectively. Taken together, basal ECV is a good predictor of CA and heart failure for MM patients.

Discovery of a New Microbial Origin Cold-Active Neopullulanase Capable for Effective Conversion of Pullulan to Panose.

Panose is a type of functional sugar with diverse bioactivities. The enzymatic conversion bioprocess to produce high purity panose with high efficiency has become increasingly important. Here, a new neopullulanase (NPase), Amy117 from B. pseudofirmus 703, was identified and characterized. Amy117 presented the optimal activity at pH 7.0 and 30 °C, its activity is over 40% at 10 °C and over 80% at 20 °C, which is cold-active. The enzyme cleaved α-1, 4-glycosidic linkages of pullulan to generate panose as the only hydrolysis product, and degraded cyclodextrins (CDs) and starch to glucose and maltose, with an apparent preference for CDs. Furthermore, Amy117 can produce 72.7 mg/mL panose with a conversion yield of 91% (w/w) based on 80 mg/mL pullulan. The sequence and structure analysis showed that the low proportion of Arg, high proportion of Asn and Gln, and high α-helix levels in Amy117 may contribute to its cold-active properties. Root mean square deviation (RMSD) analysis also showed that Amy117 is more flexible than two mesophilic homologues. Hence, we discovered a new high-efficiency panose-producing NPase, which so far achieves the highest panose production and would be an ideal candidate in the food industry.

Structural insights into xylanase mutant 254RL1 for improved activity and lower pH optimum.

Xylanases degrade xylan to valuable end products. In our previous study, the alkaline xylanase S7-xyl from Bacillus halodurans S7 was engineered by rational design and the best mutant xylanase 254RL1 exhibited 3.4-fold improvements in specific activity at pH 9.0. Further research found that the enzyme activity at pH 6.0 was almost 2-fold than that at pH 9.0. To elucidate the reason of enhanced performance of 254RL1 at decreased pH optimum, we determined the X-ray crystal structure of 254RL1 at 2.21 Å resolution. The structural analysis revealed that the mutations enlarged the opening of the access tunnel and shortened the tunnel. Moreover, the mutations changed the hydrogen bond network around the catalytic residue and decreased the pKa value of acid-base catalyst E159 which reduced the pH optimum of the xylanase. The result provided the basis for the acid-alkaline engineering of the glycoside hydrolases.

Expression of Tspan29 in breast cancer tissues and its effect on malignant biological behaviors of MCF-7 and MDA-MB-231 cells / 中国肿瘤生物治疗杂志

@#Objective: To investigate the expression of tetraspanins-29 (Tspan29) in breast cancer tissues and cell lines and to explore the effect of Tspan29 knockdown on proliferation, invasion, migration and epithelial-mesenchymal transition (EMT) of breast cancer MCF-7 and MDA-MB-231 cells. Methods:Atotal of20pairsofbreast cancer tissues and corresponding para-cancerous tissues resected in Minhang Branch of Cancer HospitalAffiliated to Fudan University from June 2017 to February 2018 were collected for this study; in addition, breast cancer celllinesMCF-7,MDA-MB-231andhumanbreastepithelialMDA-kb2cellswerealsocollected.ThemRNAand protein expressions of Tspan29 in above mentioned tissues and cell lines were detected by Real-time quantitative (qPCR) and Western blotting. The expression of Tspan29 in MCF-7 and MDA-MB-231 cells was interfered by siRNA. qPCR was used to detect the mRNA and protein expressions of Tspan29. PCR microarray was used to examine the expressions of EMT-related genes in MCF-7 cells. CCK-8 assayandTranswellwereusedtodetectcellproliferation, migration and invasion of MCF-7 and MDA-MB-231 cells. Results: The mRNA and protein expressions of Tspan29 in breast cancer tissues were significantly higher than that in para-cancerous tissues (all P<0.01); and the mRNA and protein expressions of Tspan29 in MCF-7 and MDA-MB-231 cells were significantly higher than that in MDA-kb2 cells (P<0.01). After being interfered with siTspan29, the mRNA and protein expressions of Tspan29 were significantly down-regulated in MCF-7 cells (all P<0.05); the proliferation, invasion and migration of MCF-7 and MDA-MB-231 cells were significantly inhibited (all P<0.05); and among the EMT-related genes, two were significantly up-regulated while 7 were down-regulated. Conclusion: Tspan29 is significantly up-regulated in breast cancer tissues and cell lines, and knockdown of Tspan29 significantly inhibits the proliferation, invasion and migration of breast cancer cells. ··

Expression, functional analysis and mutation of a novel neutral zearalenone-degrading enzyme.

The crops and grains were often contaminated by high level of mycotoxin zearalenone (ZEN). In order to remove ZEN and keep food safe, ZEN-degrading or detoxifying enzymes are urgently needed. Here, a newly identified lactonohydrolase responsible for the detoxification of ZEN, annotated as Zhd518, was expressed and characterized. Zhd518 showed 65% amino acid identity with Zhd101, which was widely studied for its ZEN-degrading ability. A detailed activity measurement method of ZEN-degrading enzyme was provided. Biochemical analysis indicated that the purified recombinant Zhd518 from E. coli exhibited a high activity against ZEN (207.0 U/mg), with the optimal temperature and pH of 40 °C and 8.0, respectively. The Zhd518 can degrade ZEN derivatives, and the specific activities against α-Zearalenol, ß-Zearalenol, α-Zearalanol and ß-Zearalanol were 23.0 U/mg, 64.7 U/mg, 119.8 U/mg and 66.5 U/mg, respectively. The active sites of Zhd518 were predicted by structure modeling and determined by mutation analysis. A point mutant N156H exhibited 3.3-fold activity against α-Zearalenol comparing to Zhd518. Zhd518 is the first reported neutral and the second characterized ZEN-degrading enzyme, which provides a new and more excellent candidate for ZEN detoxifying in food and feed industry.

MELK as a potential target to control cell proliferation in triple-negative breast cancer MDA-MB-231 cells.

Maternal embryonic leucine zipper kinase (MELK) is an important regulator in tumorigenesis of human breast cancer, and if silenced leads to programmed cell death in specific breast cancer cell lines, including MDA-MB-231 cells. In the present study, RNA interference, proliferation assay and semi-quantification of cell cycle relative proteins were performed to determine the effects of MELK in human breast cancer cells. Data demonstrated that the highest level of MELK protein in the MDA-MB-231 cell line among eight breast cancer cell lines. The sensitivity of MELK small interfering-RNA varied in different breast cancer cell lines, but MELK silencing resulted in marked suppression of proliferation of triple-negative breast cancer (TNBC) and non-TNBC cells. Specific silencing of MELK caused G2 arrest in TNBC MDA-MB-231 and HCC1143 cells, and G1 arrest in non-TNBC T47D and MCF7 cells. Notably, the knockdown of MELK did not induce apoptosis in HCC1143 cells, indicated by the lack of caspase-3 expression. In addition, in response to MELK silencing, cyclin B and cyclin D1 were downregulated in four breast cancer cell lines. Furthermore, the silencing of MELK resulted in the upregulation of p21, p27 and phosphorylated (p)-c-Jun N-terminal kinase (JNK) in HCC1143 TNBC cells, and downregulation of p21 and p-JNK in T47D non-TNBC cells. Additionally, MELK protein was markedly suppressed in non-TNBC cells in response to estrogen deprivation. The findings from the present study suggested that MELK may be a potential target in MDA-MB-231 cells, although genetic knockdown of MELK resulted in inhibitory effects on proliferation of TNBC and non-TNBC cells. MELK exert its effect on different breast cancer cells via arrest of different cell cycle phases and therefore mediated by different mediators, which may be involved in the crosstalk with MELK signaling and with the estrogen receptor signaling pathway.

I-7ab inhibited the growth of TNBC cells via targeting HDAC3 and promoting the acetylation of p53.

Triple negative breast cancer (TNBC) is a heterogenous disease with high aggressive and poor outcome. The lack of biomarkers and targeted therapies makes it a challenge for the treatment of TNBC. Histone deacetylase inhibitors (HDACis) are emerging as novel anti-tumor agents in many types of human cancers. In this study, we found that I-7ab, a novel HDACi, inhibited the cell viability of TNBC cells and induced the cell apoptosis. Mechanistically, I-7ab specifically decreased the expression of HDAC3 and promoted the acetylation of p53 at both Lys373 and Lys382 amino acids. The up-regulated acetylation of p53 promoted the transcriptional activity of p53 and induced the expression of p21, which consequently caused cell cycle arrest at G1 phase. Administration of I-7ab inhibited the colony formation of TNBC cells. Collectively, these results indicated I-7ab as a promising anti-cancer agent in the treatment of TNBC.

Loss of FOXN3 in colon cancer activates beta-catenin/TCF signaling and promotes the growth and migration of cancer cells.

Aberrant activation of beta-catenin/TCF is a hallmark of colon cancer. How the functions of nuclear localized beta-catenin are regulated is not fully understood. Here, it was found that FOXN3 (Forkhead box N3) was down-regulated in colon cancer tissues. Forced expression of FOXN3 inhibited the growth, migration and invasion of colon cancer cells, while knocking down the expression of FOXN3 promoted the growth, migration, invasion and metastasis of colon cancer cells. FOXN3 bind to beta-catenin and inhibited beta-catenin/TCF signaling by blocking the interaction between beta-catenin and TCF4. Taken together, these data demonstrated the suppressive roles of FOXN3 in the progression of colon cancer, and indicated that restoring the functions of FOXN3 would be a novel therapeutic strategy for colon cancer.

A core collection and mini core collection of Oryza sativa L. in China.

The extent of and accessibility to genetic variation in a large germplasm collection are of interest to biologists and breeders. Construction of core collections (CC) is a favored approach to efficient exploration and conservation of novel variation in genetic resources. Using 4,310 Chinese accessions of Oryza sativa L. and 36 SSR markers, we investigated the genetic variation in different sized sub-populations, the factors that affect CC size and different sampling strategies in establishing CC. Our results indicated that a mathematical model could reliably simulate the relationship between genetic variation and population size and thus predict the variation in large germplasm collections using randomly sampled populations of 700-1,500 accessions. We recommend two principles in determining the CC size: (1) compromising between genetic variation and genetic redundancy and (2) retaining the main types of alleles. Based on the most effective scheme selected from 229 sampling schemes, we finally developed a hierarchical CC system, in which different population scales and genetic diversities allow a flexible use of genetic resources. The CC, comprising 1.7% (932) of the accessions in the basic collection, retained more than 85% of both the SSR and phenotypic variations. A mini core collection, comprising 0.3% (189) of the accessions in the basic collection, retained 70.65% of the SSR variation and 76.97% of the phenotypic variation, thus providing a rational framework for intensive surveys of natural variation in complex traits in rice genetic resources and hence utilization of variation in rice breeding.

Genetic structure and differentiation of Oryza sativa L. in China revealed by microsatellites.

China is one of the largest centers of genetic diversity of Oryza sativa L. in the world. Using a genetically representative primary core collection of 3,024 rice landraces in China, we analyzed the genetic structure and intraspecific differentiation of O. sativa, and the directional evolution of SSR. The genetic structure was investigated by model-based structure analysis and construction of neighbor-joining phylogenetic tree. Comparison between genetic structure and predefined populations according to Ting's taxonomic system revealed a hierarchical genetic structure: two distinct subspecies, each with three ecotypes and different numbers of geo-ecogroups within each ecotype. Two subspecies evidently resulted from adaptation to different environments. The different cropping systems imposed on the subspecies led to further differentiation, but the variation within each subspecies resulted from different causes. Indica, under tropical-like or lowland-like environments, exhibited clear differentiation among seasonal ecotypes, but not among soil-watery ecotypes; and japonica showed clear differences between soil water regime ecotypes, but not among seasonal ecotypes. Chinese cultivated rice took on evident directional evolution in microsatellite allele size at several aspects, such as subspecies and geographical populations. Japonica has smaller allele sizes than indica, and this may partly be the result of their different domestication times. Allele size was also negatively correlated with latitude and altitude, and this may be interpreted by different mutation rates, selection pressures, and population size effects under different environments and cropping systems.

Assessing indica-japonica differentiation of improved rice varieties using microsatellite markers.

To assess the indica-japonica differentiation of improved rice varieties, a total of 512 modern varieties including 301 indica and 211 japonica accessions were analyzed using 36 microsatellites. The Fst coefficients ranged from 0.002 to 0.730 among the loci with an average of 0.315. Significant differentiation was detected at 94.4% of the loci studied (P < 0.05, pairwise Fst tests), indicating that there was a high level of indica-japonica differentiation within the improved varieties. At 18 loci, about 74%-98% of the alleles of indica and japonica accessions were distributed in two ranges of amplicon length. Linkage disequilibrium analysis showed that the distribution trends were significantly nonrandomly associated. Using the differentiation trends at the 18 loci, microsatellite index (MI) was proposed for discrimination of the two subspecies. When rice accessions with MI value greater than zero were classified as indica, and those with MI value smaller than zero were classified as japonica, about 96.1% of the accessions could be classified. This result agrees with the classification based on morphological-physiological characters, indicating that this method is feasible and effective.

Genetic structure and phylogeography of rice landraces in Yunnan, China, revealed by SSR.

Yunnan Province is one of the largest centers of genetic diversity of Oryza sativa L. in China, and in the world. Using a genetically representative core collection of 692 rice landraces from Yunnan, the genetic structure, differentiation, and geographic diversity of this rice germplasm were analyzed. The accessions were classified into different populations, according to the model-based structure analysis. Model-based populations were characterized through the reconstruction of a neighbor-joining tree, principal coordinate analysis, and the estimation of morphologic and SSR variations. Finally, the distribution of genetic diversity and differentiation among districts were studied. Seven model-based populations were identified on the basis of the structure analysis. This classification was partly consistent with Ting's 5-level taxonomic system. Differentiation between 2 rice subspecies is the most apparent, with a clearer differentiation between soil-watery ecotypes in japonica than in indica; however, differentiation among seasonal ecotypes in indica is clearer than in japonica. Cropping system and man-made restricted-growth environments could be considered to be the main forces driving the intraspecific differentiation of cultivated rice. It has been suggested that, because it possesses the highest genetic diversity and all the necessary conditions as a center of genetic diversity, the southwest region of Yunnan, encompassing Simao, Lincang, and Xishuangbanna districts, is the center of genetic diversity of Yunnan rice landraces.

[Genetic diversity of common wild rice (Oryza rufipogon Griff) by using SSR markers and phenotypic traits in Guangxi Province].

In this paper, allelic variation and genetic diversity among geographies and growth habit types were studied by using 223 accessions of common wild rice primary core collection in Guangxi Province, with 34 SSR primers locating on 12 chromosomes of rice and 19 phenotypic traits. In the results, 24.91 alleles were detected per locus on average with a range from 7 to 48. Compared to the cultivated rice, the wild rice showed more allelic variations. The ratio of heterozygote of SSR locus was 32.01% on average, and it's range was 1.35% 81.31%. The frequency of heterozygote of SSR locus in Oryza rufipogon Griff was much higher than in Oryza sativa L. The geographical distribution of genetic diversity measured by SSR markers was not completely accordant with that by phenotypic traits. At DNA level, more wild rice individuals and higher genetic diversity were included within the area covering north latitude 22 degrees - 23 degrees and 23 degrees - 24 degrees (comprising Longan, Fusui, Yongning, Xiangzhou, Laibin, Xuanwu, Yulin and Guigang county), which formed the center of genetic diversity. But the center of genetic diversity at the phenotypic level located within north latitude 21 degrees - 22 degrees and 22 degrees - 23 degrees. Among the four growth habit types, the genetic diversity from high to low was found respectively in prostrate type, sloping type, slighting type, and erect type at both DNA and phenotypic levels.